Project Summary There has been a tremendously increasing interest in the use of small animals such as zebrafish in drug discovery. A number of recent studies have proposed the use of zebrafish in drug discovery workflows, where they could be used to test drug candidates prior to being tested on expensive mammalian models. One significant advantage that zebrafish brings, is the ability to test entire compound libraries in a high throughput screening (HTS) fashion, a task not easily accomplished using conventional mammalian models. Using zebrafish in drug discovery could therefore decrease the chances of drug candidate failures in the preclinical stage itself. Despite these advantages, tools that allow HTS in zebrafish are not established. Most commonly used devices (96-well plates) that were designed specifically for cell- and enzyme-based screening, suffer from a number of artifacts when applied to motile organisms such as zebrafish. Microscale technologies has been shown to have potential to alleviate some of these difficulties. However, these technologies are complex and require precise fluidic control, issues which hamper their commercial adoption. In this proposal, we present the use of our novel and easy-to-use millifluidic device that can efficiently handle, load, align and manipulate zebrafish embryos. Our proposed device will streamline zebrafish based screening, and will be amenable to automation and mass production because of simple yet robust design. In order to adapt our technology to screen zebrafish, we propose the following Specific Aims: (1) Develop and optimize an integrated millifluidic device for consistent loading, dechorionation, mounting, healthy culture, and establish imaging procedures for zebrafish embryos; and (2) Test and compare feasibility of drug screening on zebrafish larvae in the millifluidic device. Robustness of our device will be tested and results will be compared with conventional well plates as reported in literature. These studies will demonstrate a proof-of-concept for the use of our device in handling and screening of zebrafish.